Structure and Localization of the Flavonoid Multienzyme Complex

生物大分子之间的相互作用是几乎每一个细胞过程的基础，包括将合作酶组织成大分子复合物。多酶系统提供了获得高局部底物浓度的手段，调节分支途径之间的竞争，协调相互依赖途径的活动，并在细胞内隔离有毒或挥发性中间体。然而，对于任何酶复合物的结构或指导单个组分相互作用的分子机制，我们所知甚少。该项目将建立在Winkel实验室先前的工作基础上，以表征拟南芥中黄酮类酶复合物的结构和功能，包括使用小角度中子散射来开发该系统的三维模型。将测试几种研究相互作用界面的新技术，以努力确认植物中提出的相互作用，并确定可能决定复合物组装的因素。最近发现的黄酮类酶的差异定位的决定因素也将被表征。这些研究将有助于了解大分子相互作用是如何以必要的特异性和灵活性发生的，以有效地介导拥挤和动态细胞内部的生物过程。这些知识对于代谢工程有效地应用于植物的农艺和营养改良以及微生物、植物和动物中有价值的代谢物的生物生产至关重要。该项目将为本科生、研究生和博士后提供当代分子遗传学、生物化学和计算生物学的培训基地。这项工作的更广泛影响还包括研究本科生研究经历对参与学生及其导师的结果和影响的具体努力。十多年来，大约35名本科生在温克尔实验室进行了研究；这为以严格和广泛适用的方式研究这些经验提供了一个理想的场景。将评估本科生在其研究经历后追求的学术和职业机会，研究经历在多大程度上准备和/或鼓励本科生追求学术和职业机会，以及本科生的研究经历对导师的影响程度。

Interactions between biological macromolecules underlie virtually every cellular process, including the organization of cooperating enzymes into macromolecular complexes. Multienzyme systems provide the means to attain high local substrate concentrations, regulate competition among branch pathways, coordinate the activities of interdependent pathways, and sequester toxic or volatile intermediates within the cell. Yet remarkably little is known about the architecture of any enzyme complex or the molecular mechanisms that direct the interaction of the individual components. The project will build on previous work in the Winkel laboratory to characterize the structure and function of the flavonoid enzyme complex in Arabidopsis thaliana, including the use of small angle neutron scattering to develop a three-dimensional model of this system. Several new technologies for studying interaction interfaces will be tested in an effort to confirm proposed interactions in planta, and to identify factors that may arbitrate assembly of the complex. The determinants of the recently discovered differential localization of flavonoid enzymes will also be characterized. These studies will contribute to a general understanding of how macromolecular interactions occur with the requisite specificity and flexibility to effectively mediate biological processes in the crowded and dynamic cell interior. This knowledge is essential for the effective application of metabolic engineering to the agronomic and nutritional improvement of plants and for the bioproduction of valuable metabolites in microorganisms, plants, and animals.The project will provide a training ground in contemporary molecular genetics, biochemistry, and computational biology for undergraduates, graduate students, and a postdoctoral fellow. The broader impact of this work also involves a specific effort to study the outcomes and impacts of research experiences for undergraduates on the participating students and their mentors. Some 35 undergraduate students have conducted research in the Winkel laboratory over more than a decade; this provides an ideal scenario for studying these experiences in a rigorous and broadly applicable way. An assessment will be made of the academic and career opportunities that undergraduates pursue following their research experience, the extent to which the research experience prepare and/or encourage undergraduates to pursue academic and career opportunities, and the extent to which research experiences for undergraduates impacted the mentors.